PROJECT SUMMARY Osteoporotic fractures occur in 1 in 3 women and 1 in 5 men over the age of 50, costing the US more than $22 billion annually. Of the six major classes of FDA-approved osteoporotic drugs, only one class works by promoting bone formation rather than blocking resorption, and it cannot be safely used for more than 2 years. Therefore, effective fracture prevention and osteoporosis treatment requires additional safe strategies to promote bone formation. In youth, a potent strategy to promote bone formation is mechanical loading, but with aging, the bone formation response to the same stimulus becomes diminished. Our long-term goal is to restore mechanoresponsiveness in aged bone to provide more strategies to treat osteoporosis and prevent fractures. A pathway essential for maximal bone formation is Wnt signaling. However, with aging, activation of Wnt signaling following loading progressively declines. Thus, enhancing Wnt signaling is a promising strategy to rescue the mechanoresponsiveness. Wnt signaling in bone can be enhanced by exogenous nerve growth factor (NGF) and by Notum inhibitors, which each target a distinct age-related deficiency. Aim 1: We propose to directly test whether mechanoresponsiveness can be restored in aged animals by treating 22-month mice with exogenous NGF or Notum inhibitors. We will evaluate bone formation following loading using dynamic histomorphometry and microCT. Wnt signaling potentiation will be assessed by Wnt target gene expression. Additional molecular pathways likely also underlie the diminished mechanoresponsiveness in aging. Preliminary bulk RNA-seq comparing the loading responses of young and old bone identified three pathways, in addition to Wnt signaling, that are enriched in young but not old mice following loading. We will extend these bulk findings with translating ribosome affinity purification (TRAP), a tool that can be used to isolate osteoblast- and osteocyte- specific transcripts. Further, some phases of mechanosensing occur only at the protein level, including degradation of the Wnt antagonist sclerostin, so we will also assess protein-level differences. Aim 2: We propose to use (1) bulk RNA-seq to compare loaded and non-loaded tibias from young and aged mice to define genes and pathways that differ with aging. We will extend our findings using (2) TRAP-seq. Last, we will use (3) proteomics to define protein-level differences in the loading response between young and aged mice. The training in this proposal will take place at the Washington University Musculoskeletal Research Center, which has a long tradition of training independent musculoskeletal researchers. This proposal takes advantage of the sponsor?s expertise using controlled in vivo loading models to study mechanoresponsiveness and Wnt signaling in mice and leverages the excellent genomics and computational resources at Washington University. Overall, the training and mentorship provided by this fellowship will facilitate the long-term goal of becoming an independent surgeon-scientist studying musculoskeletal aging.